The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning.

All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate that is hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys "catalytic triad." In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzyme is maintained despite the mutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu-59) in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 O(ε1) in the wild-type enzyme and interacts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 O(δ1.) In neither case is an atom located at the position of Glu-142 O(ε2) in the wild-type enzyme. The active site cysteine of the E142L mutant was found to form a Michael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 O(ε2) and the substrate amino group in positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.

Hypolithic microbial communities: between a rock and a hard place.

Drylands are the largest terrestrial biome on Earth and a ubiquitous feature is desert pavement terrain, comprising rocks embedded in the mineral soil surface. Quartz and other translucent rocks are common and microbial communities termed hypoliths develop as biofilms on their ventral surfaces. In extreme deserts these represent major concentrations of biomass, and are emerging as key to geobiological processes and soil stabilization. These highly specialized communities are dominated by cyanobacteria that support diverse heterotrophic assemblages. Here we identify global-scale trends in the ecology of hypoliths that are strongly related to climate, particularly with regard to shifts in cyanobacterial assemblages. A synthesis of available data revealed a linear trend for colonization with regard to climate, and we suggest potential application for hypoliths as 'biomarkers' of aridity on a landscape scale. The potential to exploit the soil-stabilizing properties of hypolithic colonization in environmental engineering on dryland soils is also discussed.

Rapid microbial response to the presence of an ancient relic in the Antarctic Dry Valleys.

The extreme cold and aridity of the Antarctic McMurdo Dry Valleys have led to the longstanding belief that metabolic rates of soil microbiota are negligible, and that ecosystem changes take place over millennia. Here we report the first direct experimental evidence that soil microbial communities undergo rapid and lasting changes in response to contemporary environmental conditions. Mummified seals, curious natural artifacts found scattered throughout Dry Valleys, alter their underlying soil environment by stabilizing temperatures, elevating relative humidity and reducing ultraviolet exposure. In a unique, multi-year mummified seal transplantation experiment, we found that endemic Dry Valley microbial communities responded to these changes within 3 years, resulting in a sevenfold increase in CO(2) flux and a significant reduction in biodiversity. These findings challenge prevailing ideas about Antarctic Dry Valley ecosystems and indicate that current and future environmental conditions may strongly influence the ecology of the dominant biota in the Dry Valleys.

Retrieval of full-length functional genes using subtractive hybridization magnetic bead capture.

Numerous gene-specific PCR methods have been developed for the cultivation-independent discovery of novel genes from complex environmental DNA samples. The recovery of full-length genes is, however, technically challenging. Here, we present an efficient and relatively simple approach that combines magnetic bead capture with subtractive hybridization for the rapid and direct recovery of full-length target ORFs. When compared with other PCR-based techniques, a higher degree of specificity is achieved through the use of larger gene fragments during hybridization followed by several high-stringency washes. Together with the recent advances in environmental nucleic acid extraction techniques, this approach should allow for the further exploration of the metagenomic sequence space.

Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8.

Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of approximately 600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, "c-shaped", circular and "figure-8" shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.

Acrolein in wine: importance of 3-hydroxypropionaldehyde and derivatives in production and detection.

Certain lactic acid bacteria strains belonging to the genus Lactobacillus have been implicated in the accumulation of 3-hydroxypropionaldehyde (3-HPA) during anaerobic glycerol fermentation. In aqueous solution 3-HPA undergoes reversible dimerization and hydration, resulting in an equilibrium state between different derivatives. Wine quality may be compromised by the presence of 3-HPA due to the potential for spontaneous conversion into acrolein under winemaking conditions. Acrolein is highly toxic and has been implicated in the development of bitterness in wine. Interconversion between 3-HPA derivatives and acrolein is a complex and highly dynamic process driven by hydration and dehydration reactions. Acrolein is furthermore highly reactive and its steady-state concentration in complex systems very low. As a result, analytical detection and quantification in solution is problematic. This paper reviews the biochemical and environmental conditions leading to accumulation of its precursor, 3-HPA. Recent advances in analytical detection are summarized, and the roles played by natural chemical derivatives are highlighted.

Hypolithic microbial community of quartz pavement in the high-altitude tundra of central Tibet.

The hypolithic microbial community associated with quartz pavement at a high-altitude tundra location in central Tibet is described. A small-scale ecological survey indicated that 36% of quartz rocks were colonized. Community profiling using terminal restriction fragment length polymorphism revealed no significant difference in community structure among a number of colonized rocks. Real-time quantitative PCR and phylogenetic analysis of environmental phylotypes obtained from clone libraries were used to elucidate community structure across all domains. The hypolithon was dominated by cyanobacterial phylotypes (73%) with relatively low frequencies of other bacterial phylotypes, largely represented by the chloroflexi, actinobacteria, and bacteriodetes. Unidentified crenarchaeal phylotypes accounted for 4% of recoverable phylotypes, while algae, fungi, and mosses were indicated by a small fraction of recoverable phylotypes.

Endolithic microbial colonization of limestone in a high-altitude arid environment.

The morphology of endolithic colonization in a limestone escarpment and surrounding rocky debris (termed float) at a high-altitude arid site in central Tibet was documented using scanning electron microscopy. Putative lichenized structures and extensive coccoid bacterial colonization were observed. Absolute and relative abundance of rRNA gene signatures using real-time quantitative polymerase chain reaction and phylogenetic analysis of environmental phylotypes were used to characterize community structure across all domains. Escarpment endoliths were dominated by eukaryotic phylotypes suggestive of lichenised associations (a Trebouxia lichen phycobiont and Leptodontidium lichen mycobiont), whereas float endoliths were dominated by bacterial phylotypes, including the cyanobacterium Chroococcidiopsis plus several unidentified beta proteobacteria and crenarchaea. Among a range of abiotic variables tested, ultraviolet (UV) transmittance by rock substrates was the factor best able to explain differences in community structure, with eukaryotic lichen phylotypes more abundant under conditions of greater UV-exposure compared to prokaryotes. Variously pigmented float rocks did not support significantly different communities. Estimates of in situ carbon fixation based upon (14)C radio-labelled bicarbonate uptake indicated endolithic productivity of approximately 2.01 g C/m(2)/year(-1), intermediate between estimates for Antarctic and temperate communities.

Thermophilic ethanologenesis: future prospects for second-generation bioethanol production.

Strategies for improving fermentative ethanol production have focused almost exclusively on the development of processes based on the utilization of the carbohydrate fraction of lignocellulosic material. These so-called 'second-generation' technologies require metabolically engineered production strains that possess a high degree of catabolic versatility and are homoethanologenic. It has been suggested that the production of ethanol at higher temperatures would facilitate process design, and as a result the engineered progeny of Geobacillus thermoglucosidasius, Thermoanerobacterium saccharolyticum and Thermoanerobacter mathranii now form the platform technology of several new biotechnology companies. This review highlights the milestones in the development of these production strains, with particular focus on the development of reliable methods for cell competency, gene deletion or upregulation.

Sources of edaphic cyanobacterial diversity in the Dry Valleys of Eastern Antarctica.

Cyanobacteria are major components of Antarctic Dry Valley ecosystems. Their occurrence in lakes and ponds is well documented, however, less is known about their distribution in edaphic environments. There has been considerable debate about the contribution of aquatic organic matter derived largely from cyanobacteria to terrestrial ecosystems. In this study, automated rRNA intergenic spacer analysis (ARISA) and 16S rRNA gene clone libraries were used to investigate cyanobacterial diversity in a range of soil environments within the Miers and Beacon Valleys. These data were used to elucidate the input of aquatic cyanobacteria to soil communities. Thirty-eight samples were collected from a variety of soil environments including dry and moist soils, hypoliths and lake and hydroterrestrial microbial mats. The results from the ARISA and 16S rRNA clone library analysis demonstrated that diverse cyanobacterial communities exist within the mineral soils of the Miers Valley. The soil samples from Beacon Valley were depauparate in cyanobacterial signals. Within Miers Valley, significant portions (29%-58%) of ARISA fragment lengths found in aquatic cyanobacterial mats were also present in soil and hypolith samples, indicating that lacustrine and hydroterrestrial cyanobacteria play a significant role in structuring soil communities. The influence of abiotic variables on the community structure of soil samples was assessed using BEST analysis. The results of BEST analysis of samples from within Miers Valley showed that total percentage of carbon content was the most important variable in explaining differences in cyanobacterial community structure. The BEST analyses indicated that four elements contributed significantly to species compositional differences between valleys. We suggest that the complete absence of lakes or ponds from Beacon Valley is a contributing factor to the low cyanobacterial component of these soils.

Structure of an aliphatic amidase from Geobacillus pallidus RAPc8.

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 A was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4(2)32. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily alpha-beta-beta-alpha fold. Association in the hexamer preserves the eight-layered alpha-beta-beta-alpha:alpha-beta-beta-alpha structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity.

The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing.

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 A resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4(2)32, with unit-cell parameter a = 130.49 (+/-0.05) A. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.

Bacterial diversity in three different Antarctic Cold Desert mineral soils.

A bacterial phylogenetic survey of three environmentally distinct Antarctic Dry Valley soil biotopes showed a high proportion of so-called "uncultured" phylotypes, with a relatively low diversity of identifiable phylotypes. Cyanobacterial phylotypic signals were restricted to the high-altitude sample, whereas many of the identifiable phylotypes, such as the members of the Actinobacteria, were found at all sample sites. Although the presence of Cyanobacteria and Actinobacteria is consistent with previous culture-dependent studies of microbial diversity in Antarctic Dry Valley mineral soils, many phylotypes identified by 16S rDNA analysis were of groups that have not hitherto been cultured from Antarctic soils. The general belief that such "extreme" environments harbor a relatively low species diversity was supported by the calculation of diversity indices. The detection of a substantial number of uncultured bacterial phylotypes showing low BLAST identities (< 95%) suggests that Antarctic Dry Valley mineral soils harbor a pool of novel psychrotrophic taxa.